We investigated the effect of a sulfhydryl-modifying reagent on insulin receptor functions in digitonin-permeabilized Chinese hamster overy cells that were transfected with human insulin receptors (IR). The regent used was maleimidobutyryl biocytin (MBB), a thiol-specific biotinylation reagent. Results showed that MBB had an enhancing effect on the insulin receptor tyrosine kinase activity toward an exogenous substrate. In the absence of insulin, MBB caused an increase in basal kinase activity comparable to the level seen with insulin alone. Treatment of permeabilized cells with MBB caused further 2-fold stimulation of receptor kinase activity by insulin. IR ~-subunit contains sulfhydryl group(s) that react readily with MBB. The extent of biotinylation of IR ~-subunit was identical whether the cells were stimulated or not with insulin. Biotinylation of ~-subunit was totally inhibited by L-cysteine but not L- glycine, indicating the specific nature of MBB as a thiol-modifying reagent. Several sulfhydryl reagents were tested as probes for assessing the location of accessible thiol(s) on the receptor as well as determining whether these reactive sulfhydryl(s) are vicinals. It appears that modification of IR sulfhydryl(s) by MBB did not cause reduced accessibility of cellular protein tyrosine phosphatases toward IR phosphorylation sites. We conclude that modification of reactive cysteine(s) by MBB results in enhanced IR tyrosine kinase activity, which could suggest an important role played by cellular oxidation of such residues in insulin signalling.